The long-term goal of this research program is to understand the functions of Ser/Thr protein kinases (STPKs) in the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Mtb causes tuberculosis (TB), and it infects one third of the world's population. TB kills ~2 million people annually, and drug resistant strains are emerging rapidly. To target the Mtb STPKs with new therapeutics, basic research is needed to define the specific biological functions and regulatory mechanisms of these enzymes. In the first grant period, we pioneered structural studies of the Mtb STPKs and established the current paradigm that dimerization and phosphorylation mediated by two structural interfaces activate bacterial STPKs. We also developed novel approaches to identify STPK substrates in Mtb. These studies afford the opportunity to focus biochemical, biophysical, structural and genetic methods to establish new principles of bacterial STPK signaling. The breadth and novelty of approaches are strengths of this program. This research has four specific aims: 1. Define the basis for signaling through Mtb transmembrane receptor kinases. 2. Discover networks of kinase cross-phosphorylation and regulation. 3. Discover candidate protein substrates of Mtb STPKs. 4. Determine how the STPKs regulate the Mtb flippase for peptidoglycan precursors. Our preliminary studies established the conceptual framework and the feasibility of these aims. We propose several groundbreaking studies, including spatial and temporal mapping of kinase localization, dimerization and activation in vivo. We will test the hypotheses that dimerization activates bacterial STPKs, that the complete Mtb kinome forms a hierarchical network, that the STPKs regulate much of Mtb physiology, and that PknB senses cell wall fragments and regulates peptidoglycan biosynthesis. Because of the central roles of kinase signaling in cellular physiology, the increasing focus on STPKs as pharmaceutical targets, and the worldwide health impact of TB, our proposed studies will have high significance for molecular biology and medicine. PUBLIC HEALTH RELEVANCE: By discovering the mechanisms of environmental sensing underlying tuberculosis (TB) and other bacterial infections, this program will connect directly to ongoing efforts to develop new antibiotics against major life-threatening diseases. Human cells also signal through similar molecular sensors, called protein kinases, and these kinases are important targets for drugs to treat diseases including diabetes, inflammation, pain and cancer. Consequently, this program has broad and direct implications for human health.